Pedigree reconstruction and genetic analysis of major ornamental characters of ornamental crabapple (Malus spp.) based on paternity analysis

Ornamental crabapple is an important woody ornamental plant in the Northern Hemisphere. Its flowers, fruits, leaves and tree habit are all important ornamental characters. As there has been no research on the selection of superior parents and phenotypic variation, new varieties of ornamental crabapple are mainly selected from open-pollination progeny. In order to explore the transmission rule of ornamental traits between parents and offspring of crabapple, and to provide a basis for the selection of hybrid parents for directional breeding, 14 pairs of SSR markers were used in this study for paternity analysis of 384 offspring from 4 female parents crossed with 91 candidate male parents. And 273 offspring (71.1%) were matched with only the father at a 95% strict confidence level. We reconstructed 7 full-sib families (number of progeny ≥ 10) on the basis of the paternity analysis results. Genetic analysis of characters in the full-sib families revealed that green leaves and white flowers were dominant traits. All the hybrid offspring from the white flower (♀) × non-white flower (♂) cross produced white flowers, while 7.04% produced non-white flowers when both parents had white flowers. The results showed that white flowers might be a dominant qualitative trait in crabapple, while the depth of red was a quantitative trait. The genetic characteristics of green and non-green leaves and the depth of red of the peel were similar to flower color. Compared with the upright and spreading traits, the weeping trait was recessive. Some progeny showed an earlier blooming period, indicating the possibility of breeding for blooming period. Our findings are important for parent screening and improving the breeding efficiency of new varieties in ornamental crabapple hybridization.

Ornamental crabapples (Malus spp.) are plants in the Rosaceae with a fruit diameter of less than 5 cm and flowers, fruits, leaves and other traits of ornamental value. As important woody ornamental plants in temperate regions of the Northern Hemisphere, they are widely used in landscaping and landscape design 1,2 . There are many varieties of ornamental crabapples. Among the more than 200 varieties reported at present, only slightly more than 60 have known parents 2 . Most of the varieties were selected from the progeny of natural hybrids, with complex genetic backgrounds and unclear genetic relationships. The breeding of crabapple varieties is still achieved by selecting the offspring of natural hybrids on the basis of excellent ornamental traits and retaining them through vegetative propagation. For example, the new cultivars M. 'FengHong NiChang' 3 and M. 'Fen Balei' 4 were selected from Malus halliana and Malus micromalus, respectively. As it is difficult to identify the male parent in this process, it is impossible to breed a large number of new varieties with high efficiency by artificial crossbreeding for traits, which restricts the directed breeding of ornamental crabapples. Our laboratory aimed to use a variety of traditional methods to conduct artificial hybridization of ornamental crabapples. Due to the low fertility of Data analysis. Genotyping results were analyzed by Peak Scanner V1.0 software (Thermo Fisher Scientific, MA, USA). The segments were arranged in order from smallest to largest as A, B, C…, the segments with the same base size were represented by the same capital letter, and missing data were indicated by '.. ' . The number of observed alleles (Na), observed heterozygosity (Ho), and Nei's diversity index was determined using POPGENE version 1.32 software 21 . Paternity analysis was performed based on the maximum likelihood method by comparing genotypes of known maternal origin and offspring against candidate paternal materials using Cervus 3.0 software 22 with a genotyping error rate set to 0.01. Table 1. Ornamental trait classification. The varieties with an early blooming period bloomed between March 31 and April 4, and those with a medium blooming period bloomed between April 5 and April 9. Varieties that entered the blooming period after April 10 were recorded as having a late blooming period, and those that still had not bloomed in May were considered non-blooming These categories were based on the blooming period of M. 'Pink Spires' (March 31), which began blooming earliest. The color of flowers, leaves and fruit was checked on the sunny side using the Royal Horticultural Society Standard Color Chart (RHSCC). The leaf base was wide, the length was approximately twice the width (ovate-round), and the length and width were similar to those of broadly ovate leaves. In contrast to ovate-round leaves, the narrow leaf base led to an obovate shape. When the middle part of the leaf is the widest and the length is approximately 1.5-2 times the width, the leaf is recorded as broadly elliptical; when the length is 3 to 4 times the leaf width, the leaf is recorded as long elliptical.  (Table 3). Since half of the genetic variation of offspring comes from only 4 mothers and half comes from the part of the 91 candidate parents. Although there were far more offspring than candidate parents, the genetic diversity of the progeny was still lower than that of the candidate parents (except for gssr-21). All the genotypes from the 14 pairs of SSR primers were available in Supplementary Information 2.
Paternity analysis of half-sib families. Of the 384 offspring from the 4 half-sib families, 273 (71.09%) were matched to a unique paternal tree at a 95% strict confidence level. The average matching success rate of the 4 families was 71.09%, with the M. 'Sweet Sugartyme' family being the highest at 75% and the M. 'Darwin' family being the lowest at 66.67% (Fig. 1a). Table S2 in Supplementary

Family reconstruction and trait variation analysis of the full-sib families.
According to the paternity analysis, we reconstructed the full-sib families of ornamental crabapples and selected 7 hybrid combinations with more than 10 progenies from these families (Table S3 in Supplementary Information 1). The characters of 7 parents are shown in Table 4. A summary of the results from flower color, leaf color, fruit color, leaf shape, fruit size and tree habit is shown in Table 5. When the flower color was divided into white and non-white (pink and red), white was dominant over non-white. The hybrid progenies of white flower (♀) × non-white flower (♂) crosses all had white flowers. When both parents had white flowers, 92.92% of the offspring had white flowers and 7.04% had non-white flowers, indicating that the production of white flowers was not recessive. The offspring with green leaves accounted for 90.76%, and only 4 red and 6 purple progenies resulted from red leaf (♀) × green leaf (♂) crosses. There were only 2 red-leaved individuals among the hybrids, and both parents had green leaves. These results prove that green leaves were dominant over red leaves. The ratio of red to non-red (yellow and green) offspring was near 1:1 in red fruit (♀) × non-red fruit (♂) crosses. The red-fruited offspring accounted for 80% when a red-fruited female was crossed with a green-fruited male. The other two combinations mainly produced non-red and red peels. Overall, the ratio of red to non-red fruits was close to 1:1, and red and yellow were the main fruit colors, accounting for 45.38% and 40.00%, respectively.
Spreading was the most common habit among the offspring. Differentiation occurred in the progeny regardless of parental tree habit. For example, most of the offspring were spreading and a few offspring were drooping www.nature.com/scientificreports/ when an upright female was crossed with a spreading male. Approximately 2% of progenies were weeping when two spreading parents were crossed. However, the proportion of spreading offspring (approximately 10%) was significantly increased if the male or female parent was drooping or weeping. Parents had similar effects on the leaf shape and fruit size of offspring in the7 full-sib families. It was rare that these two traits in offspring deviated significantly from those of the parents. Table 6 shows the results for four traits, including leaf surface, glossiness of skin, fruit calyx, and blooming period. The leaf surface of offspring was affected by the parents; most progenies were similar to the parents, but   www.nature.com/scientificreports/ a few were differentiated (transgressive inheritance). There were two types of strongly expressed and weakly expressed glossiness of skin in parents. The phenotypes of offspring were mostly within the range of values observed for the parents, but some progenies did not display one of the parents' phenotypes.
Only two types of blooming periods were observed for the parents and offspring, early and medium, with 80.00% of progeny showing the medium type. Similarly, the fruit calyx trait had only two states: absent and always present. The fruit calyx was absent in 63.85% of progenies, regardless of parental phenotype.

Discussion
Genetic diversity is not only an important index used to measure species' ability to adapt to changing environments but also a key factor affecting plant genetic improvement 23 . Studying the genetic diversity of ornamental crabapple varieties provides a molecular basis for hybridization selection. Hokanson et al. 24 used 8 pairs of SSR primers to analyze the genetic diversity of 142 Malus plants, and the average He was 0.623. Kumar et al. 25 used SSR molecular markers to analyze the genetic diversity of wild crabapple populations in the Himalayan region of India, and the He value was 0.506. In this study, the average He of 91 ornamental crabapple varieties was 0.81, which was significantly higher than the previously reported values. On the one hand, the 91 parental varieties   www.nature.com/scientificreports/ in this study were from a wide range of sources, and compared with that of wild populations, their genetic background was complex, so the diversity level was higher. On the other hand, the number of markers used in this study was higher than that used in previous studies, and the capillary electrophoresis technology was more accurate. Moreover, the 384 progenies of the 4 families also had a high level of genetic diversity (He = 0.76), but the level was lower than that of the candidate parents (0.81). The main reason was that not all candidate parents provided pollen, and the paternal parents of these progeny came from only 44 varieties. Paternity analysis has been extensively used in various plant studies, such as paternity testing, pedigree reconstruction, mating system examination and dynamic changes in genetic diversity in generations. Ai et al. 26 used 11 pairs of SSR primers to analyze 286 seeds from a Pinus massoniana seed orchard with 129 candidate paternal clones, and paternity at a 95% and an 80% confidence level was determined for 25 seeds (8.80%) and 107 seeds (37%), respectively. However, in a small candidate parent population of Moringa oleifera with only 60 male parents, 8 pairs of SSR primers assigned fathers for 155 of 288 seeds (53.82%) based on a 95% strict confidence level 27 . By comparing the results of the two studies, it can be seen that the number of candidate parents 28 and polymorphism of the SSR molecular markers used are the main factors that affect the accuracy and efficiency of paternity analysis. Furthermore, the sampling intensity of candidates is another important factor in paternity analysis. In this study, 91 candidate paternal parents were screened through phenological observations. Using 14 pairs of SSR primers at a 95% confidence level, 273 offspring (71.1%) were matched. It was obvious that using capillary electrophoresis to replace polyacrylamide gel electrophoresis was also an important way to improve efficiency and accuracy. Further analysis of the distance between the males and females in the nursery showed that M. 'Red Sentinel' and M. 'Winter Red' , the male parents that produced the most offspring, were located far from the female parents, indicating no obvious relationship between pollination success rate and the distance of pollen transmission. The compatibility of male and female gametes might be the key factor affecting the pollination success rate. M. 'Red Sentinel' and M. 'Winter Red' have good compatibility with most varieties when used as male or female parent and are suitable as parents for hybrid experiments.
In this study, the genetic characteristics of traits in parents and offspring were analyzed through reconstructed full-sib families to provide a basis for parental selection in crossbreeding. Flower color is one of the main ornamental traits of crabapple, but there are no reports on the genetics of this trait in ornamental crabapple. According to genetic studies on the flower color of Camellia azalea Wei and Hibiscus coccineus Walter, white flowers are recessive to red flowers 29,30 . However, Han et al. 31 found that the petal color of F 1 offspring derive from the cross between cabbage with yellow petals (♀) and Chinese kale with white petals (♂) was white, and the segregation conformed to a Mendelian ratio of 3:1 in F 2 offspring originating from self-pollination of F 1 plants, proving that white petals were dominant over yellow petals. In the natural population of ornamental crabapples examined in this study, the flowers were mostly white. If flower color was divided into white and non-white (pink and red), the offspring from the cross between parents with white flowers (♀) and non-white flowers (♂) all had white flowers, indicating that white flowers were dominant over non-white flowers. When both parents had white flowers, only 7.04% of the offspring had non-white flowers, which again indicated that the white flower trait was dominant. Of course, the results need to be confirmed by crossing two parents with red flowers. In addition, the intensity of red was a continuous trait showing quantitative characteristics, and the molecular mechanism underlying its formation needs to be further studied.
The accumulation of anthocyanins causes red leaves in many crops and ornamental plants. Previous studies proved that the inheritance of red or purple leaves followed a monogenic recessive pattern 32,33 . In contrast, red or purple leaves are controlled by a single dominant gene in birch, copper beech and Brassica juncea [34][35][36] . Huang et al. 37 found that leaf color was determined by a single locus and that the purple leaf phenotype was recessive to the green leaf phenotype by selfing Kalanchoe garambiensis (purple leaves) and K. garambiensis G. (green leaves). Our study showed that 97.53% of the offspring resulting from crosses between parents with green leaves also produced green leaves. The green leaf trait was observed among 79.59% of offspring from red (♀) × green (♂) crosses. This finding suggests that green leaves were dominant over red or purple leaves. However, the segregation ratio of green and red leaves in offspring deviated from that expected under Mendelian inheritance, which might be related to the quantity of offspring.
Previous studies on apples found that fruit color was composed of background and surface color. Red was dominant over yellow and controlled by the single gene Rf [38][39][40] . However, some scholars believe that the inheritance of skin color is regulated by major genes as well as polygenes. Although red is dominant, the intensity of red is affected by polygenes. It is a qualitative trait with a quantitatively inherited character 41 . In our study, the progenies with red fruits accounted for 80% of the progenies obtained from red (♀) × green (♂) crosses, and the segregation ratio of red and yellow was close to 1:1 in some full-sib families. We speculated that red peel was also dominant in crabapple.
Tree habit is a major ornamental trait controlled by both major genes and polygenes 42 . It has been proven that the weeping trait is regulated by single recessive genes in peach and Canadian redbud 33,43 . There were not only major pl genes but also some polygenes involved in controlling the weeping trait in Prunus mume 44 . In our data, upright and spreading were the main tree habits in offspring, and progenies rarely exhibited drooping and weeping. A few drooping and weeping progenies appeared in the crosses of upright (♀) × spreading (♂) and spreading (♀) × spreading (♂), respectively. The proportion of drooping and weeping in offspring was significantly increased if a parent was drooping or weeping. The results showed that weeping and drooping were recessive. However, the segregation ratio of two full-sib families' offspring deviated from that expected under Mendelian inheritance, and we speculated that the production of weeping branches in crabapple may be similar to that in P. mume. Dougherty et al. 45 revealed four genomic regions, W, W2, W3 and W4, that were significantly associated with weeping by performing genetic mapping in the F 1 generation of a cross between Malus 'Cheal's Weeping' and Malus 'Evereste' , and W was the major locus, which further supported the inference of this study. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.